C-Terminal Hsp90 Inhibitors Overcome MEK and BRAF Inhibitor Resistance in Melanoma

Abstract

Targeted therapies for melanoma MEK and BRAF inhibitors fail due to the development of chemoresistance. As Hsp90 inhibitors target client proteins of resistant pathways, we hypothesised that C-terminal Hsp90 inhibitors will target BRAF/MEK inhibitor resistant melanoma cells by overcoming the resistant pathways. Two melanoma cell lines, A375 and A375 MEK/BRAF inhibitor resistant (A375MEKi) were utilised. The inhibitory concentrations (IC₅₀) of two C-terminal Hsp90 inhibitors, KU757 and KU758, were determined by CellTiter Glo. RNA sequencing was performed after treatment with KU757. Pathways targeted by differentially expressed genes were evaluated by David, IPA, GSEA, and by evaluating the cell cycle, apoptosis and oxidative phosphorylation. Expression levels of hub genes were evaluated using Xena and validated by RT-PCR. The survival analysis was performed using UALCAN. A375MEKi was not resistant to the C-terminal Hsp90 inhibitor with a KU757 IC₅₀ of 0.59 μM versus 0.64 μM and a KU758 IC₅₀ of 0.89 μM versus 0.93 μM in A375 versus A375MEKi, respectively. RNA sequencing analysis revealed KU757 upregulates cell cycle checkpoint regulation and apoptosis and downregulates genes involved in the peroxisome, AKT/PI3K/MTOR, EIF2, fatty acid metabolism and oxidative phosphorylation. These pathways were further validated through survival analysis that demonstrated potential survival benefit in patients with dysregulated NDUFA7, CDC20, CDC25C, CDK1, VDAC2, HEATR5a, COL4A4, FLT3LG, BMP2, PRKCH and ADMST9. Melanomas often develop concurrent resistance to BRAF and MEK inhibitors. C-terminal Hsp90 inhibition with KU757 appears to overcome these chemo-resistance pathways in vitro, downregulating metabolic pathways including oxidative phosphorylation and the cell cycle, warranting further in vivo translation. The novel C-terminal HSP90 inhibitor KU757 effectively targets primary and BRAF and MEK inhibitor-resistant melanoma cells equally by affecting oxidative phosphorylation and the cell cycle.